Hemophilia is a bleeding disorder caused by a hereditary genetic mutation on the X chromosome that leads to a deficiency of a blood coagulation factor. Coagulation is a process of stopping blood loss from a damaged blood vessel wherein the damaged blood vessel wall is covered by a fibrin-containing clot formed through a complex coagulation cascade associated with various coagulation factors. Of the coagulation factors, factor VIII (herein referred to as FVIII) and factor IX (FIX) are associated with the onset of hemophilia A and B, respectively, when they are deficient.
Hemophilia B occurs when FIX is so deficient or inactive that the coagulation cascade for clot formation does not take place. To treat hemophilia B, FIX is administered in various amounts depending on the level of coagulation factors and the type of hemorrhage.
For use in the treatment of hemophilia B, FIX may be typically produced by two methods: purification from human blood; and genetic recombination. A recombinant protein, although producible in a large amount, is poorer in activity and stability than a protein obtained by plasma fractionation.
Various attempts including random mutagenesis, structure-activity relationship comparison, PEGylation, and n-glycosylation have been made on recombinant proteins to overcome the disadvantages, but most of them have failed to achieve special effects.
Transferrin is a blood plasma protein that transports iron through the blood. This plasma protein is the third most abundant in the blood, has a half-life of 8 days, which is relatively long although shorter than that of albumin or immunoglobulin G (IgG), and is featured by receptor-mediated circulation. There have been several fusion proteins that employ transferrin as a fusion partner, but neither the use of transferrin in fusion to FIX nor an effect thereof has been found in any report ever published.
Therefore, the present inventors have endeavored to improve the activity and stability of FIX; and have found that when linked directly or via a linker to transferrin, FIX was notably increased in specific activity and blood stability, compared to non-fused, native FIX, and thus accomplished the present invention.